Estrogen receptors (ER) are expressed in ∼65% of human breast cancer. Cumulative data from clinical trials and retrospective analyses suggest that some chemotherapeutic agents may be less effective in patients with ER-positive (ER+) tumors than those with ER-negative (ER−) tumors. Paclitaxel is an active agent used in breast cancer chemotherapy. To investigate the possible influence of ER on the therapeutic efficacy of paclitaxel and its underlying mechanism, we established several isogenic ER+ cell lines by stable transfection of ERα expression vectors into ER− breast cancer BCap37 cells. We showed that 17-β estradiol significantly reduces the overall cytotoxicity of paclitaxel in BCap37-expressing ERα but has no influence on the ER− parental cells. Further analyses indicate that expression of ERα in BCap37 cells mainly interferes with paclitaxel-induced apoptotic cell death, without affecting paclitaxel-induced microtubule bundling and mitotic arrest. Moreover, we found that the addition of ICI 182,780 (Fulvestrant), a selective ER down-regulator, could completely reverse the resistance of ER+ BCap37 cells to paclitaxel. These findings showed that ERα-mediated breast tumor cell resistance to paclitaxel was through selective inhibition of paclitaxel-induced tumor cell apoptosis. Additionally, the combination of ICI 182,780 also sensitizes MCF-7 and T47D cell lines to the treatment of paclitaxel, which further confirmed the correlation between ERα and drug resistance in ER+ tumor cells. The results obtained from this study provide useful information for understanding ER-mediated resistance to paclitaxel and possibly other antineoplastic agents. [Cancer Res 2007;67(11):5337–44]

Estrogen receptors (ER) are transcriptional factors that play an important role in the development and progression of breast cancers (13). Cumulative analysis of tumor biopsies has shown that ERs are present in ∼65% of human breast tumors (4). It has long been known that breast cancers that express the ERα protein (ER+) behave in a fundamentally different fashion than ER-negative (ER−) tumors with regard to their response to hormonal therapies (46). In recent years, data from clinical trials or retrospective analyses suggested that ER status might also affect the efficacy of chemotherapy (710). Specifically, it has been observed that some chemotherapeutic agents may be less effective in patients with ER+ tumors than those with ER− tumors. These findings indicate that ER status may play an important role in determining the sensitivity of breast tumors to chemotherapy, although the mechanisms underlying ER-mediated drug resistance are not entirely clear (8, 1113).

Taxanes (paclitaxel and docetaxel), a novel class of naturally occurring antimicrotubule agents, represent active chemotherapeutic agents developed in the last two decades for the treatment of malignancies, including breast cancer (14, 15). However, not all breast tumors are sensitive to taxanes. Evidence is accumulating that improvements in taxane-based adjuvant chemotherapy disproportionately benefit patients with ER− breast tumors (9, 16, 17). Indeed, such a finding has also been observed in our in vitro experiments (18). In earlier studies, we characterized the possible correlation of the activation of nuclear factor-κB (NF-κB)/IκB pathway with the susceptibility of tumor cells to paclitaxel-induced apoptosis (1820). We found that the breast cancer cell line MCF-7 was highly resistant to paclitaxel-induced apoptosis although it was still responsive to paclitaxel in microtubule bundling and mitotic arrest (18). However, we did not appreciate that this resistance might be associated with ER status. Recently, we found that both paclitaxel-sensitive cell lines (human breast cancer cell line BCap37 and ovarian cancer cell line OV2008) used in this comparative study are ER− (see Fig. 1A). This finding raised the possibility that the insensitivity of MCF-7 cell line to paclitaxel-induced apoptosis might be correlated to its ER.

Figure 1.

ERα expression attenuates the overall cytotoxicity of paclitaxel. A, whole cellular protein extracts of BCap37 cells transfected with empty vector or ERα were analyzed by Western blot with an anti-ERα antibody as described in Materials and Methods. T47D and MCF-7 cells were used as positive controls of ERα expression. B, MTT assays. Cells cultured in 96-well microplates were treated with 1 nmol/L 17-β estradiol, 50 nmol/L paclitaxel, or the combination treatment in which cells were preincubated with 17-β estradiol for 12 h before paclitaxel treatment. Cell viability was evaluated by MTT assays after both 48 and 72 h of paclitaxel treatment. Columns, mean of three independent experiments; bars, SE. BC-V, BCap37 transfected with empty vector; BC-ER, pooled transfectants of BCap37 transfected with ERα; BC-ER1–7, single clones 1 to 7 of BCap37 transfected with ERα; CTL, control; EST, 17-β estradiol; PTX, paclitaxel. #, P < 0.05, when compared with the group treated with paclitaxel alone in the same cell line; *, P < 0.001, when compared with the group treated with paclitaxel alone in the same cell line.

Figure 1.

ERα expression attenuates the overall cytotoxicity of paclitaxel. A, whole cellular protein extracts of BCap37 cells transfected with empty vector or ERα were analyzed by Western blot with an anti-ERα antibody as described in Materials and Methods. T47D and MCF-7 cells were used as positive controls of ERα expression. B, MTT assays. Cells cultured in 96-well microplates were treated with 1 nmol/L 17-β estradiol, 50 nmol/L paclitaxel, or the combination treatment in which cells were preincubated with 17-β estradiol for 12 h before paclitaxel treatment. Cell viability was evaluated by MTT assays after both 48 and 72 h of paclitaxel treatment. Columns, mean of three independent experiments; bars, SE. BC-V, BCap37 transfected with empty vector; BC-ER, pooled transfectants of BCap37 transfected with ERα; BC-ER1–7, single clones 1 to 7 of BCap37 transfected with ERα; CTL, control; EST, 17-β estradiol; PTX, paclitaxel. #, P < 0.05, when compared with the group treated with paclitaxel alone in the same cell line; *, P < 0.001, when compared with the group treated with paclitaxel alone in the same cell line.

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Therefore, in this study, we determined whether the differential sensitivity of breast cancer cells to paclitaxel might be a consequence of ERα expression. Through stable transfection of an ERα expression vector into ER− BCap37 cells, we found that, in the presence of 17-β estradiol, the expression of ERα in BCap37 cells is clearly associated with decreased sensitivity to paclitaxel. Analyses including DNA fragmentation, flow cytometry, and terminal deoxynucleotidyl transferase–mediated nick end labeling (TUNEL) assays indicate that 17-β estradiol significantly interferes with the ability of paclitaxel to induce apoptosis in BCap37 cells transfected with ERα, but not in parental ER− BCap37 cells. Moreover, ICI 182,780 (Fulvestrant), a steroidal pure antiestrogen agent (21, 22), dramatically down-regulates ERα protein levels and nearly completely reverses the resistance to paclitaxel in BCap37 cells transfected with ERα. Meanwhile, the combination of ICI 182,780 was also found to sensitize ER+ MCF-7 and T47D cells to paclitaxel, which provided additional evidence that expression and subsequent activation of ERα are associated with resistance to paclitaxel in breast cancer cells.

Cell culture and agents. Human breast cancer cell lines BCap37 (19, 20), BCap37 transfected with pIRES-ERα expression vector (BC-ER), or empty vector (BC-V) were maintained in RPMI 1640, whereas MCF-7 and T47D cells were cultured in DMEM supplemented with 10% fetal bovine serum. Paclitaxel and 17-β estradiol were purchased from Sigma and dissolved in 100% DMSO and ethanol, respectively. ICI 182,780 was purchased from Tocris and dissolved in DMSO. Cells were cultured in phenol-free medium containing 10% dextran-coated, charcoal-treated FCS (Hyclone) before they were treated with 17-β estradiol, paclitaxel, and ICI 182,780 alone or in combination.

Stable transfection and selection of ERα-transfected cells. The pIRES expression vector containing a cytomegalovirus promoter and the pIRES-ERα expression vector have been used in our previous studies (23, 24). Transfection was done by Lipofectin (Life Technologies) as recommended by the manufacturer. Briefly, ER− BCap37 cells cultured in 6-cm dishes were washed twice and supplemented with 3 mL Opti-MEM reduced serum medium. pIRES-ERα or pIRES plasmid DNA (2 μg per 6-cm dish) was mixed with Lipofectin before addition to tumor cells. After transfection, stable transfectants were selected by incubating with 500 μg/mL geneticin (G418). Surviving colonies were picked ∼2 weeks later. Single colonies were amplified and examined for ERα expression by Western blotting. Positive clones were maintained in culture medium supplemented with 250 μg/mL G418.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Drug-induced cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays as described previously (19). Briefly, tumor cells were evenly cultured into 96-well tissue culture plates for approximately overnight and then treated with designated drugs. At the end of experiments, MTT solution was added into the 96-well plates and the plates were incubated for additional 3 h, allowing viable cells to reduce the yellow tetrazolium salt (MTT) into dark blue formazan crystals. Finally, DMSO was added to dissolve the formazan crystals. The absorbance in individual wells was determined at 562 nm by a microplate reader.

Detection of internucleosomal DNA fragmentation. After exposure to treatments, both parental BCap37 and ERα-transfected clones were harvested and suspended in lysis solution for 30 min on ice. Crude DNA samples were extracted twice with phenol/chloroform/isoamyl alcohol (25:24:1). The remaining steps for detection of DNA fragmentation were done as described previously (25). DNA samples were analyzed by electrophoresis in a 1.5% agarose slab gel containing 0.2 μg/mL ethidium bromide and visualized under UV illumination.

TUNEL assay. The TUNEL assay was carried out using the method previously described (2628). Briefly, cells were trypsinized and affixed to slides by using a Cytospin 3 (Shandon) cell preparation system after various treatments. The slides were air dried and fixed with 4% paraformaldehyde, followed by TUNEL staining according to the manufacturer's protocol of a commercial kit (Roche). 3,3′-Diaminobenzidine was used as a substrate for signal conversion (28). Apoptotic cells were identified by positive TUNEL staining and five randomly selected microscopic fields in each group were used to calculate the relative ratio of TUNEL-positive cells.

Flow cytometric analysis. Cell cycle distribution and apoptosis was further determined by flow cytometric analysis. Cell sample preparation was done as described previously (29). Briefly, at the end of each time point, cells were harvested and fixed in 70% ethanol diluted in PBS. Cells were then incubated in PBS containing 100 μg/mL RNase and 40 μg/mL propidium iodide at room temperature for 0.5 to 1.0 h before flow cytometry analysis. Cell cycle distribution and DNA content were determined using a Coulter Epics V instrument with an argon laser set to excite at 488 nm. The results were analyzed using Elite 4.0 software (Phoenix Flow System).

Cytospin preparation. After designated treatments, BCap37 cells with or without expression of ERα were harvested by trypsinization and washed with PBS. Approximately 0.5 × 105 to 1 × 105 cells were plated onto microscope slides using the Cytospin 3 cell preparation system. As described previously (30), slides were air dried and fixed in absolute methanol before Giemsa staining. Slides from three independent experiments were examined and photographed using bright-field microscopy. Five randomly selected microscopic fields with at least 500 cells were counted to calculate the percentage of mitotic cells in each group. Only those cells with typical morphologic features of condensed chromosomes were counted as mitotically arrested cells.

Immunofluorescence staining of microtubules. Cells were allowed to adhere to Lab-Tek II chamber glass slides overnight and subsequently treated with 17-β estradiol and paclitaxel as described above. After 24 h of paclitaxel treatment, cells were gently washed with PBS and fixed in 100% methanol for 10 min. Cells were permeabilized for 10 min with 0.2% Triton X-100, followed by blocking at room temperature for 30 min in 5% bovine serum albumin. After washing thrice with PBS, cells were stained with FITC-conjugated anti–α-tubulin monoclonal antibody (Clone DM1A, Sigma) in a dark chamber for 1 h at room temperature (18, 31). Cells were washed with PBS before mounting and imaged with Zeiss LSM 510 confocal microscope.

Western blotting. Cellular protein was isolated with a protein extraction buffer containing 150 mmol/L NaCl, 10 mmol/L Tris (pH 7.2), 5 mmol/L EDTA, 0.1% Triton X-100, 5% glycerol, and 1% SDS. Equal amounts (40 μg/lane) of proteins were fractionated on 10% to 12% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. The membranes were incubated with anti-ERα (Santa Cruz Biotechnology), IκBα (Santa Cruz Biotechnology), bcl-2 (DAKO), c-raf (BD Biosciences), and cyclin B1 (Santa Cruz Biotechnology) primary antibodies, respectively. After washing with PBS containing 0.1% (v/v) Tween 20, the membranes were incubated with peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratories) followed by enhanced chemiluminescent staining using the ECL system (Amersham Biosciences).

Statistical analysis. Data are presented as mean ± SE of three independent experiments. Student's t test was used to determine the statistical difference between various experimental and control groups. Differences were considered statistically significant at a level of P < 0.05.

Stable transfection and expression of ERα in BCap37 cells. The human beast cancer BCap37 cell line has been used as a well-characterized cell model in our previous studies for the evaluation of taxane-containing combination therapy (19, 20, 3032). This ER− cell line is very sensitive to paclitaxel and docetaxel in both mitotic arrest and apoptosis. To investigate the possible influence of ER status on the sensitivity of breast cancer cells to paclitaxel or possibly other chemotherapeutic agents, we restored expression of ERα in BCap37 cells through stable transfection of ERα expression vector (pIRES-ERα). Western blotting confirmed the expression of exogenous ERα in clones transfected with pIRES-ERα cDNA. As shown in Fig. 1A, the BCap37 cell line transfected with empty vector does not express ERα (lane 1, BC-V), whereas the pooled ERα transfectants (lane 8, BC-ER) and individual ERα-transfected clones 1, 2, 3, 5, and 6 (lanes 2, 3, 4, 6, and 7), express high levels of exogenous ERα that are similar to T47D and MCF-7 cells (lanes 9 and 10), two recognized ERα-positive human breast cancer cell lines (33). Clone 4 does not express detectable ERα protein (lane 5) and was not used for further studies.

BCap37 cells expressing ERα exhibit reduced sensitivity to paclitaxel. Next, we did MTT assays to determine whether the expression of ERα would affect the sensitivity of BCap37 cells to paclitaxel. Briefly, the cells transfected with empty vector (BC-V), pooled transfectants (BC-ER), and three selected ERα-positive individual clones (BC-ER1, BC-ER3, and BC-ER6) were treated with 50 nmol/L paclitaxel with or without pretreatment of 1 nmol/L 17-β estradiol administered 12 h before paclitaxel treatment. MTT assays were done after cells were exposed to paclitaxel for 48 and 72 h, respectively. As depicted in Fig. 1B, we found that 1 nmol/L 17-β estradiol alone showed little effect on cell viability in all cell lines tested. In the absence of 17-β estradiol, ERα-transfected cell lines exhibited similar (BC-ER, BC-ER1, and BC-ER3) or slightly decreased (BC-ER6) sensitivity to paclitaxel compared with the vector-transfected cells (BC-V). However, in the presence of 17-β estradiol, the cell viabilities after paclitaxel treatment were much higher in all cell lines transfected with ERα than that of the cell line transfected with vector only (P < 0.05 or P < 0.001; see Fig. 1B). These results indicate that, in the presence of 17-β estradiol, the overall cytotoxic effects of paclitaxel were significantly reduced in the BCap37 cells expressing ERα.

Expression of ERα represses paclitaxel-induced apoptosis. Next, we examined the possible inhibitory effect of ERα on the ability of paclitaxel to induce tumor cell apoptosis. DNA fragmentation assays were done after BC-V cells, BC-ER1, and BC-ER6 clones were exposed to paclitaxel for 48 h. As shown in Fig. 2A, the characteristic DNA fragmentation ladders were observed in all three cell lines treated with 50 nmol/L paclitaxel alone (lanes 4, 8, and 12). Pretreatment with 17-β estradiol did not affect paclitaxel-induced apoptosis in BC-V cells (Fig. 2A, lane 5), but it significantly attenuated paclitaxel-induced DNA fragmentation ladders in both clones expressing ERα (lanes 9 and 13). Further, TUNEL assay was carried out to evaluate the frequency of apoptosis. As shown in Fig. 2B, there was no significant difference in the percentage of TUNEL-positive cells between groups exposed to paclitaxel alone or the combination with 17-β estradiol and paclitaxel in BC-V cells (18.07% versus 16.33%, P > 0.05). However, there were significantly fewer TUNEL-positive cells in the group cotreated with 17-β estradiol than those treated with paclitaxel alone in BC-ER6 cells (19.72% versus 8.69%, P < 0.001). Moreover, the flow cytometric analysis also indicates that in BC-ER6 cell line, the apoptotic peak (Fig. 3, AP) induced by paclitaxel alone was much higher than that of cotreatment with 17-β estradiol (particularly at 72 h), but estrogen has little effect on paclitaxel in the parental BC-V cells. These results show that 17-β estradiol markedly inhibited paclitaxel-induced apoptotic cell death in BCap37 cells expressing exogenous ERα.

Figure 2.

Expression of ERα interferes with paclitaxel-induced apoptosis. Apoptotic cell death was determined by DNA fragmentation and TUNEL assays when tumor cells treated with 1 nmol/L 17-β estradiol, 50 nmol/L paclitaxel, or their combination for 48 h. A, DNA fragmentation assay. B, representative results and quantitative analysis of TUNEL staining. TUNEL-positive cells were shown as brown staining under the light microscope. Columns, mean TUNEL labeling percentage based on five randomly selected high-power microscopic fields for each group; bars, SE. Bar, 100 μm for all photographs. *, P < 0.001, when compared with the group treated with paclitaxel alone in the same cell line.

Figure 2.

Expression of ERα interferes with paclitaxel-induced apoptosis. Apoptotic cell death was determined by DNA fragmentation and TUNEL assays when tumor cells treated with 1 nmol/L 17-β estradiol, 50 nmol/L paclitaxel, or their combination for 48 h. A, DNA fragmentation assay. B, representative results and quantitative analysis of TUNEL staining. TUNEL-positive cells were shown as brown staining under the light microscope. Columns, mean TUNEL labeling percentage based on five randomly selected high-power microscopic fields for each group; bars, SE. Bar, 100 μm for all photographs. *, P < 0.001, when compared with the group treated with paclitaxel alone in the same cell line.

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Figure 3.

Flow cytometric analyses of cell cycle distribution and apoptosis. Cells treated with 1 nmol/L 17-β estradiol, 50 nmol/L paclitaxel, or their combination for indicated time points were harvested, and DNA content were stained with propidium iodide for flow cytometric analysis. Peaks corresponding to G1, G2-M, and S phases of the cell cycle and apoptotic cells (AP).

Figure 3.

Flow cytometric analyses of cell cycle distribution and apoptosis. Cells treated with 1 nmol/L 17-β estradiol, 50 nmol/L paclitaxel, or their combination for indicated time points were harvested, and DNA content were stained with propidium iodide for flow cytometric analysis. Peaks corresponding to G1, G2-M, and S phases of the cell cycle and apoptotic cells (AP).

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Expression of ERα has little influence on paclitaxel-induced mitotic arrest. In our previous studies, we found that glucocorticoids could selectively inhibit paclitaxel-induced apoptosis without interfering with paclitaxel-mediated mitotic arrest (19, 31, 34, 35). To investigate whether a similar effect is also observed for ER-mediated resistance to paclitaxel, we compared the influence of preincubation with 17-β estradiol on the ability of paclitaxel to arrest tumor cells at G2-M phase in BCap37 cells with or without expression of ERα. From the flow cytometry results presented in Fig. 3, we observed that, after 48 h of paclitaxel treatment, the majority of tumor cells were accumulated at G2-M phase in both BC-V and BC-ER6 cell lines. Although preincubation with 17-β estradiol has little effect in BC-V cell line, it significantly decreases the population of cells at G2-M phase in the BC-ER6 cell line compared with the treatment of paclitaxel alone. Instead, most of the tumor cells remain at G1 phase of the cell cycle. Similar results were obtained after 72 h of paclitaxel treatment, which was accompanied with a decreased apoptotic population of tumor cells in BC-ER6 cells. These data indicate that 17-β estradiol attenuates paclitaxel-induced G2-M arrest in BCap37 cells transfected with ERα. However, because flow cytometric assay could not distinguish cells at G2 phase from M phase, we were unable to determine whether estrogen prevents tumor cells from entering G2 phase or M phase, or both. To clarify this issue, we prepared cytospin slides in which the mitotically arrested cells were easily identified by their morphologic features (e.g., condensed chromosomes; ref. 30). Through bright-field microscopy, we found that, compared with BC-ER6 cells treated with paclitaxel alone, the number of cells arrested at the mitotic phase was only slightly less when cells were preincubated with estrogen (48.93% versus 44.31%, P > 0.05; Fig. 4A). We estimate that such a marginal alteration in the cell numbers arrested at mitotic phase was caused by the ability of estrogen to increase the G1 population rather than affect the cytotoxic effect of paclitaxel on mitotic arrest.

Figure 4.

ERα status has little influence on paclitaxel-induced mitotic arrest and microtubule bundling. Cells were treated with 1 nmol/L 17-β estradiol, 50 nmol/L paclitaxel, or the combination treatment in which cells were preincubated with 17-β estradiol for 12 h before paclitaxel treatment. A, after 24 h of paclitaxel exposure, cells were harvested and affixed to slides by cytospin centrifugation and subjected to Giemsa staining. Slides were examined and photographed with a light microscope. The mitotically arrested cells exhibit the typical morphologic feature of condensed chromosomes. Bar, 100 μm. B, cells were fixed and the microtubules were visualized by immunofluorescence staining using an anti–α-tubulin antibody. Bar, 50 μm. Inset, ×2 digital zoom of area marked with asterisk.

Figure 4.

ERα status has little influence on paclitaxel-induced mitotic arrest and microtubule bundling. Cells were treated with 1 nmol/L 17-β estradiol, 50 nmol/L paclitaxel, or the combination treatment in which cells were preincubated with 17-β estradiol for 12 h before paclitaxel treatment. A, after 24 h of paclitaxel exposure, cells were harvested and affixed to slides by cytospin centrifugation and subjected to Giemsa staining. Slides were examined and photographed with a light microscope. The mitotically arrested cells exhibit the typical morphologic feature of condensed chromosomes. Bar, 100 μm. B, cells were fixed and the microtubules were visualized by immunofluorescence staining using an anti–α-tubulin antibody. Bar, 50 μm. Inset, ×2 digital zoom of area marked with asterisk.

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Expression of ERα does not affect paclitaxel-induced microtubule bundling. To further determine the possible influence of ERα on the antimicrotubule activity of paclitaxel, we did immunofluorescence staining to determine whether the expression of ERα could directly affect the ability of paclitaxel to alter microtubule structures. BC-V and BC-ER6 cells cultured on glass slides were treated with 50 nmol/L paclitaxel in the presence or absence of 1 nmol/L 17-β estradiol. After 24 h of paclitaxel incubation, the microtubule network was visualized by immunofluorescence staining with an anti–α-tubulin antibody. As shown in Fig. 4B, in control and 17-β estradiol-treated groups, most cells had a fine microtubule network that excluded the nucleus and extended throughout the cytoplasm. However, treatment with paclitaxel in BC-V and BC-ER6 cells enhanced microtubule polymerization as visualized by distinctive rigidity, increased density of cellular microtubules, and formation of microtubule bundles around the nucleus. However, the morphology of microtubule structures in cells cotreated with 17-β estradiol and paclitaxel was quite similar to that treated with paclitaxel alone in both BC-V and BC-ER6 cell lines. These data suggest that the expression of ERα has little influence on the cytotoxic effect of paclitaxel on microtubules and mitotic arrest.

Expression of ERα alters expression of apoptosis-associated proteins. The data mentioned above reveal that stable transfection and expression of ERα result in reduced sensitivity of BCap37 cells to paclitaxel, particularly to paclitaxel-induced apoptotic cell death. To investigate the potential mechanisms and genes that may be involved in these processes, we examined several regulatory proteins associated with the G2-M phase of the cell cycle and paclitaxel-induced apoptosis. The results depicted in Fig. 5 indicate that several apoptosis associated proteins exhibit differential responses to paclitaxel in BC-ER cells. We first analyzed the possible alterations of IκBα and bcl-2 that were previously reported to play important roles in paclitaxel-induced apoptosis (19, 20, 36, 37). Estrogen alone has no effect on either IκBα or bcl-2 expression. As reported previously (19, 30), paclitaxel causes bcl-2 phosphorylation and degradation of IκBα. However, in BC-ER cells, pretreatment with 17-β estradiol attenuated the paclitaxel-induced bcl-2 phosphorylation and IκBα down-regulation. The responses of c-raf to paclitaxel and 17-β estradiol treatment are quite similar to bcl-2. Additionally, we also examined the expression of cyclin B1, a G2-M checkpoint protein (30, 38). The results indicated that the protein levels of cyclin B1 were increased in response to paclitaxel, but there was no significant difference in the cells with or without expression of ERα.

Figure 5.

Western blot analyses for the ERα, IκBα, bcl-2, c-raf, and cyclin B1 proteins. Cells were treated with 1 nmol/L 17-β estradiol, 50 nmol/L paclitaxel, or the combination treatment in which cells were preincubated with 17-β estradiol for 12 h before paclitaxel treatment. Whole-cell proteins were extracted from cells after 24 h of paclitaxel treatment. Equal amounts (40 μg/lane) of cellular protein were fractionated on 10% to 12% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes, followed by immunoblotting with anti-ERα, IκBα, bcl-2, c-raf, and cyclin B1 antibodies. β-Actin protein was blotted as a control.

Figure 5.

Western blot analyses for the ERα, IκBα, bcl-2, c-raf, and cyclin B1 proteins. Cells were treated with 1 nmol/L 17-β estradiol, 50 nmol/L paclitaxel, or the combination treatment in which cells were preincubated with 17-β estradiol for 12 h before paclitaxel treatment. Whole-cell proteins were extracted from cells after 24 h of paclitaxel treatment. Equal amounts (40 μg/lane) of cellular protein were fractionated on 10% to 12% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes, followed by immunoblotting with anti-ERα, IκBα, bcl-2, c-raf, and cyclin B1 antibodies. β-Actin protein was blotted as a control.

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ICI 182,780 sensitizes ER+ breast cancer cells to paclitaxel. To further confirm the direct correlation between ERα and the resistance to paclitaxel in breast cancer cells, we studied whether “knockdown” of ERα would abrogate the resistance to paclitaxel. ICI 182,780, a novel specific ER down-regulator that is currently in clinical practice (21, 22), was used to answer this question. First, parental BC-V cells and cells transfected with ERα were treated with 100 nmol/L ICI 182,780, 1 nmol/L 17-β estradiol, and 50 nmol/L paclitaxel, alone or in combinations. MTT assays and DNA fragmentation were done to evaluate the cell viability and the sensitivity to paclitaxel-induced apoptosis, respectively. As indicated in Fig. 6, pretreatment with ICI 182,780 completely prevents 17-β estradiol-induced resistance to paclitaxel as well as paclitaxel-induced DNA fragmentation in BC-ER6 cells (Fig. 6A). In BC-V cell line, neither ICI 182,780 nor its combination with 17-β estradiol, had significant effect on overall cytotoxicity or induction of DNA fragmentation. Moreover, we showed that the treatment with ICI 182,780 significantly down-regulated the protein levels of ERα in BC-ER6 cells (Fig. 6B,, lanes 58 in panel 1). Similar to the results shown in Fig. 5, pretreatment with 17-β estradiol attenuated the paclitaxel-induced down-regulation of IκBα (Fig. 6B,, lane 3 in panel 2). Interestingly, the addition of ICI 182,780 effectively restored the sensitivity of BC-ER6 cells to paclitaxel-induced IκBα degradation in the presence of 17-β estradiol (Fig. 6B,, lane 8 in panel 2). Next, MCF-7 and T47D cell lines were used to determine whether knockdown of endogenous ERα would also increase the sensitivity of breast cancer cells to paclitaxel. Our data indicate that the pretreatment with 1 μmol/L ICI 182,780 for 12 h significantly sensitizes both MCF-7 and T47D cells to paclitaxel (Fig. 6C, P < 0.01 at both concentrations of paclitaxel). Similar to the phenomena observed in BC-ER cells (Fig. 6B), ICI 182,780 effectively down-regulates the protein levels of ERα in MCF-7 and T47D cells (Fig. 6D,, lanes 3, 4, 7, and 8 in panel 1), as well as enhanced paclitaxel-induced down-regulation of IκBα (Fig. 6D , lanes 8 and 9 in panel 2). These results indicate that down-regulation of ERα protein by ICI 182,780 significantly reverses ER-mediated resistance and produces an additive or even synergistic effect in ER+ breast tumor cells treated with paclitaxel.

Figure 6.

ICI 182,780 abrogates the resistance of ERα-positive tumor cells to paclitaxel. As described in Materials and Methods, BCap37 cells were treated with 1 nmol/L 17-β estradiol, 50 nmol/L paclitaxel, 100 nmol/L ICI 182,780, or their various combinations. MCF-7 and T47D cells were treated with 100 nmol/L 17-β estradiol, 500 or 2,000 nmol/L paclitaxel, 1 μmol/L ICI 182,780, or their various combinations. Columns, mean of three independent experiments; bars, SE. A, determination of cell viability by MTT assays and apoptosis by DNA fragmentation assays after BC-V and BC-ER cells were exposed to paclitaxel for 48 h. B, effect of 17-β estradiol, paclitaxel, ICI 182,780, and their various combinations on the expression of ERα and IκBα in BC-ER cells. C, determination of cell viability after MCF-7 and T47D cells were exposed to paclitaxel for 72 h. D, effect of 17-β estradiol, paclitaxel, ICI 182,780, and their various combinations on the expression of ERα and IκBα in T47D cells. Proteins were extracted from cells after 24 h of paclitaxel treatment. β-Actin protein was blotted as a control.

Figure 6.

ICI 182,780 abrogates the resistance of ERα-positive tumor cells to paclitaxel. As described in Materials and Methods, BCap37 cells were treated with 1 nmol/L 17-β estradiol, 50 nmol/L paclitaxel, 100 nmol/L ICI 182,780, or their various combinations. MCF-7 and T47D cells were treated with 100 nmol/L 17-β estradiol, 500 or 2,000 nmol/L paclitaxel, 1 μmol/L ICI 182,780, or their various combinations. Columns, mean of three independent experiments; bars, SE. A, determination of cell viability by MTT assays and apoptosis by DNA fragmentation assays after BC-V and BC-ER cells were exposed to paclitaxel for 48 h. B, effect of 17-β estradiol, paclitaxel, ICI 182,780, and their various combinations on the expression of ERα and IκBα in BC-ER cells. C, determination of cell viability after MCF-7 and T47D cells were exposed to paclitaxel for 72 h. D, effect of 17-β estradiol, paclitaxel, ICI 182,780, and their various combinations on the expression of ERα and IκBα in T47D cells. Proteins were extracted from cells after 24 h of paclitaxel treatment. β-Actin protein was blotted as a control.

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Drug resistance is one of the major obstacles limiting the success of cancer chemotherapy. Biological mechanisms contributing to drug resistance may be present de novo or arise after exposure to anticancer drugs (39, 40). In this field, the most widely studied phenomenon is multidrug resistance that has been linked to overexpression of a membrane associated P-glycoprotein (39). In recent years, both clinical observations and experimental studies suggested that steroid hormones and their receptors might also affect the therapeutic efficacy of antineoplastic drugs (713). For example, our previous studies showed that glucocorticoids, such as dexamethasone, could significantly interfere with the antitumor activities of paclitaxel in vitro and in vivo (19, 28, 31, 35). In the present study, we investigated the potential influence of ERα on the therapeutic effects of paclitaxel and its underlying mechanisms. Through stable transfection of an ERα expression vector into ERα-negative BCap37 cells, we have successfully established several individual ER+ BCap37 cell lines. Subsequently, we showed that restoration of ERα expression in ER− tumor cells could significantly reduce the sensitivity of tumor cells to paclitaxel. The results presented above show that ERα mainly interferes with the cytotoxic effects of paclitaxel on apoptotic cell death, although it also slightly affects paclitaxel-induced G2-M arrest due to the ERα-mediated increase in the G1 phase. These findings suggest that ER status might play an important role in determining the sensitivity of breast tumors to paclitaxel and possibly other chemotherapeutic agents.

Paclitaxel is a naturally occurring antimitotic agent that is active in the treatment of a variety of human solid tumors, including breast cancer (14, 15). However, not all breast tumors are sensitive to paclitaxel. Cumulative data from clinical observations and retrospective analyses suggested that ER status might affect the therapeutic efficacy of paclitaxel because it has been observed that paclitaxel exhibited less effect in patients with ER+ breast tumors than those with ER tumors (7, 9, 16). Moreover, the phenomenon of ER-mediated resistance to paclitaxel has also been observed in in vitro experiments (17, 41). However, most of these results were obtained based on comparative studies between the tumor cell lines derived from different individuals (17). Although some paired cell lines were derived under the selective pressure of a low/no estrogen environments, these tumor cells are still not likely to be isogenic because many features, including their proliferative capacity, might have changed due to genetic alterations (41). Thus, it is difficult to elucidate the cellular and molecular mechanisms. Therefore, the pairs of isogenic breast cell lines generated by stable transfection of ERα or empty vector may provide us a valuable model system to investigate the mechanism underlying ERα-mediated breast tumor cell resistance to paclitaxel and possibly other chemotherapeutic agents.

Our previous studies found that glucocorticoids selectively inhibited paclitaxel-induced apoptosis without affecting the ability of paclitaxel to induce microtubule bundling and G2-M arrest in breast cancer cells (31, 34, 35). This finding implies that apoptotic cell death induced by paclitaxel may occur via a signaling pathway independent of microtubule and G2-M arrest (19, 35). In the present study, we did a series of assays to examine whether the ER-mediated tumor cells resistance to paclitaxel is due to a similar mechanism. By DNA fragmentation and TUNEL assays, we determined that, similar to glucocorticoids, estrogen could significantly interfere with the ability of paclitaxel to induce apoptosis in BCap37 cells expressing exogenous ERα (Fig. 2). However, unlike with glucocorticoids, the proportion of the cell population at G2-M phase in the ER+ cells exposed to paclitaxel and estrogen seemed to be less than that of the cells treated with paclitaxel alone (Fig. 3). This phenomenon raises a question of whether estrogen can also affect the cytotoxic effect of paclitaxel on microtubule bundling and mitotic arrest. Thus, we did immunofluorescence staining to determine whether estrogen can affect the cytotoxic effect of paclitaxel on microtubules. Interestingly, we found that estrogen had marginal effect on microtubule dynamics in the cells treated with paclitaxel (Fig. 4B), but may decrease the G2-M population through the increase of cells at the G1 phase (Fig. 3), which is similar to the phenomena observed by Zajchowski et al. (42). Further, we did cytospin assays and confirmed that estrogen has little effect on mitotic arrest (Fig. 4A). These interesting results suggest that reactivation of the ER pathway may interrupt specific downstream events of paclitaxel-induced mitotic arrest, or inhibition of paclitaxel-induced apoptosis by activation of ERα may occur via a pathway independent of mitotic arrest. However, the question still remains whether G1 arrest and decreased G2-M arrest by estrogen may affect paclitaxel-induced apoptosis. Further studies are required to elucidate this issue.

To investigate the possible molecular mechanisms by which ERα mediates resistance to paclitaxel, we have examined several genes or regulatory proteins that may contribute to paclitaxel activity in inducing cell cycle arrest and apoptosis. Our data indicate that paclitaxel caused bcl-2 and c-raf phosphorylation, and IκBα degradation in both BC-V and BC-ER cell lines (Fig. 5). However, the pretreatment with 17-β estradiol could inhibit all of the above alterations induced by paclitaxel in BC-ER cells, although it has no effect in BC-V cells (Fig. 5). IκBα is the specific cytoplasmic inhibitory protein of NF-κB. The degradation of IκBα may result in the activation of NF-κB (43). Our recent studies have suggested that activation of the NF-κB/IκBα signaling pathway plays an active role in mediating paclitaxel-induced apoptosis (1820). Interestingly, the addition of the antiestrogen agent ICI 182,780 could completely reverse the above ERα-mediated inhibition of paclitaxel-induced IκBα degradation (Fig. 6B). These results may explain in part the interference of ERα with paclitaxel-induced apoptosis and suggest that the NF-κB/IκBα signaling pathway may play an active role in this process. In addition, it is well known that cyclin B1 is periodically expressed in the cell cycle and accumulates in the G2-M phase (30, 38). Our results indicate that paclitaxel causes increased levels of cyclin B1 in both BC-V and BC-ER cells, which coincides with paclitaxel-induced G2-M arrest irrespective of the ER status. When cells were pretreated with 17-β estradiol before paclitaxel, the up-regulation of cyclin B1 was still present, consistent with the data obtained from cytospin and microtubule staining showing that ERα expression has little effect on paclitaxel-induced mitotic arrest. These findings provide additional insights into the molecular mechanism of ERα-mediated resistance to paclitaxel in breast cancer cells.

ICI 182,780 is an effective ER antagonist that reduces cellular levels of ERs (21, 22). To further confirm the finding that ERα mediates resistance to paclitaxel, we examined whether the knockdown of ERα by ICI 182,780 would resensitize BC-ER, MCF-7, and T47D cells to paclitaxel. The results show that the addition of ICI 182,780 dramatically reverses the resistance of these tumor cells to paclitaxel (Fig. 6). These results provided additional evidence for the correlation between ERα and the resistance to paclitaxel in breast cancer cells. Because ICI 182,780 has been successfully used in the treatment of ER+ advanced breast cancers, our experimental results also provide evidence for the clinical strategy to combine ICI 182,780 and paclitaxel in the treatment of ER+ breast tumors. Additionally, we found that this pure antiestrogen agent has more activity in reversing 17-β estradiol-induced resistance to paclitaxel in BC-ER cells, when compared with tamoxifen, a selective ER modulator (data not shown).

In summary, the present study has investigated the possible influence of ERα on the therapeutic effects of paclitaxel on breast cancer cells. Through establishment of several isogenic cell lines by stable transfection of ERα expression vectors into ER− breast cancer cells, we have shown that 17-β estradiol significantly reduces the overall cytotoxicity of paclitaxel in BCap37-expressing ERα. Further analyses indicate that expression of ERα in BCap37 cells mainly interferes with paclitaxel-induced apoptotic cell death, and has little effect on the ability of paclitaxel to induce microtubule bundling and mitotic arrest. Additionally, we found that the addition of ICI 182,780, a selective ER inhibitor that can down-regulate the ERα protein levels, could completely reverse the ER-mediated resistance and sensitize ER+ BCap37, MCF-7, and T47D cells to paclitaxel. Put together, these findings suggest that ER status might play an important role in determining the sensitivity of breast cancer to paclitaxel and possibly other chemotherapeutic agents. The results obtained from this study may provide useful information for understanding ER-mediated resistance to paclitaxel and improving the clinical application of this class of antineoplastic agents.

Grant support: NIH grants CA92280 (W. Fan), CA88843 (N.E. Davidson), and CA109274 (B.H. Park).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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